Electrospinning can be used to prepare nanofiber mats from diverse polymers and polymer blends with embedded metallic, ceramic, or other types of nanoparticles. Such nanofiber mats have been investigated for diverse applications, such as filtration, batteries, and supercapacitors; food packaging; or biomedicine and biotechnology. A large area of research is the application of nanofibrous membranes in tissue engineering. Typically, the cell adhesion and proliferation as well as the viability of mammalian cells are tested by seeding the cells on the substrates under examination, cultivating them for a defined period of time, and finally dyeing them to enable differentiation between cells and substrates under a white light or fluorescence microscope. While this procedure works well for cells cultivated in well plates or petri dishes, other substrates may undesirably also be colored by the dye. Here, we show investigations of the optical contrast between dyed CHO DP-12 (Chinese hamster ovary) cells and different nanofiber mats electrospun from pure poly(acrylonitrile) (PAN) as well as PAN/gelatin, PAN/keratin, and PAN/TiO₂. After cultivation for 5 days, the cells were fixed with glyoxal and afterwards dyed with haematoxylin–eosin (H&E), PromoFluor 488 premium, 4,6-diamidino-2-phenylindole (DAPI), or Hoechst 33342. Examination by white light or fluorescence microscopy, respectively, revealed that, in particular, the PAN/gelatin nanofiber mats—which were the most advantageous regarding cell growth—were also colored by the dyes. This paper reports the optimized dyeing parameters to maximize the optical contrast between CHO cells and nanofiber mats.